Polypeptides

ABSTRACT

The present invention relates to polypeptides, and nucleic acids DNA encoding these polypeptides, capable of eliciting an immune reaction against cancer, methods for generating T lymphocytes capable of recognising and destroying tumour cells, and pharmaceutical compositions for the treatment, prophylaxis or diagnosis of cancer.

The present invention relates to polypeptides, and nucleic acids DNAencoding these polypeptides, capable of eliciting an immune reactionagainst cancer, methods for generating T lymphocytes capable ofrecognising and destroying tumour cells, and pharmaceutical compositionsfor the treatment, prophylaxis or diagnosis of cancer.

Cancer develops through a multistep process involving several mutationalevents. These mutations result in altered expression/function of genesbelonging to two categories: oncogenes and tumour suppressor genes.Oncogenes arise in nature from proto-oncogenes through point mutationsor translocations, thereby resulting in a transformed state of the cellharbouring the mutation. Oncogenes code for and function through aprotein. Proto-oncogenes are normal genes of the cell which have thepotential of becoming oncogenes. In the majority of cases,proto-oncogenes have been shown to be components of signal transductionpathways. Oncogenes act in a dominant fashion. Tumour-suppressor geneson the other hand, act in a recessive fashion, i.e. through loss offunction, and contribute to oncogenesis when both alleles encoding thefunctional protein have been altered to produce non-functional geneproducts.

In the field of human cancer immunology, the last two decades have seenintensive efforts to characterise genuine cancer specific antigens. Inparticular, effort has been devoted to the analysis of antibodies tohuman tumour antigens. The prior art suggests that such antibodies canbe used for diagnostic and therapeutic purposes, for instance inconnection with an anti-cancer agent. However, antibodies can only bindto tumour antigens that are exposed on the surface of tumour cells. Forthis reason, the effort to produce a cancer treatment based on theimmune system of the body has been less successful than anticipated.

A fundamental feature of the immune system is that it can distinguishself from nonself molecules and that it does not normally react againstself molecules. It has been shown that rejection of tissues or organsgrafted from other individuals is an immune response to the foreignantigens on the surface of the grafted cells. The immune responsecomprises a humeral response, mediated by antibodies, and a cellularresponse. Antibodies are produced and secreted by B lymphocytes, andtypically recognise free antigen in native conformation. They cantherefore potentially recognise almost any site exposed on the antigensurface. In contrast to antibodies, T cells, which mediate the cellulararm of the immune response, recognise antigens only in the context ofmajor histocompatability complex (MHC) molecules, and only afterappropriate antigen processing. This antigen processing usually consistsof proteolytic fragmentation of the protein, resulting in polypeptidesthat fit into the groove of the MHC molecules. This enables T cells toalso recognise polypeptides derived from intracellular proteinfragments/antigens.

T cells can recognise aberrant polypeptides derived from anywhere in thetumour cell, in the context of MHC molecules on the surface of thetumour cell. The T cells can subsequently be activated to eliminate thetumour cell harbouring the aberrant polypeptide. In experimental modelsinvolving murine tumours it has been shown that point mutations inintracellular “self” proteins may give rise to tumour rejectionantigens, consisting of polypeptides differing in a single amino acidfrom the normal polypeptide. The T cells recognising these polypeptidesin the context of MHC molecules on the surface of the tumour cells arecapable of killing the tumour cells and thus rejecting the tumour fromthe host (Boon et al., 1989, Cell 58: 293–303).

MHC molecules in humans are normally referred to as HLA (human leukocyteantigen) molecules. There are two principal classes of HLA molecules:class I and class II. HLA class I molecules are encoded by HLA A, B andC subloci and primarily activate CD8+ cytotoxic T cells. HLA class IImolecules, on the other hand, primarily activate CD4+ (cytotoxic orhelper) T cells, and are encoded by the HLA DR, DP and DQ subloci. Everyindividual normally has six different HLA class I molecules, usually twoalleles from each of the three subgroups A, B and C, although in somecases the number of different HLA class I molecules is reduced due tothe occurrence of the same HLA allele twice. For a general review, seeRoitt, I. M. et al. (1998) Immunology, 5^(th) Edition, Mosby, London.

The HLA gene products are highly polymorphic. Different individualsexpress distinct HLA molecules that differ from those found in otherindividuals. This explains the difficulty of finding HLA matched organdonors in transplantations. The significance of the genetic variation ofthe HLA molecules in immunobiology lies in their role as immune-responsegenes. Through their polypeptide binding capacity, the presence orabsence of certain HLA molecules governs the capacity of an individualto respond to specific polypeptide epitopes. As a consequence, HLAmolecules influence resistance or susceptibility to disease.

T cells may inhibit the development and growth of cancer by a variety ofmechanisms. Cytotoxic T cells, both HLA class I restricted CD8+ and HLAclass II restricted CD4+, may directly kill tumour cells presenting theappropriate tumour antigens. Normally, CD4+ helper T cells are neededfor cytotoxic CD8+ T cell responses, but if the polypeptide antigen ispresented by an appropriate APC, cytotoxic CD8+ T cells can be activateddirectly, which results in a quicker, stronger and more efficientresponse.

In International Application PCT/N092/00032 (published as WO92/14756),synthetic polypeptides and fragments of oncogene protein products whichhave a point of mutation or translocations as compared to theirproto-oncogene or tumour suppressor gene protein are described. Thesepolypeptides correspond to, completely cover or are fragments of theprocessed oncogene protein fragment or tumour suppressor gene fragmentas presented by cancer cells or other antigen presenting cells, and arepresented as a HLA-polypeptide complex by at least one allele in everyindividual. The polypeptides were shown to induce specific T cellresponses to the actual oncogene protein fragment produced by the cellby processing and presented in the HLA molecule. In particular, it isdescribed in WO92/14756 that polypeptides derived from the p21-rasprotein which had point mutations at particular amino acid positions,namely positions 12, 13 and 61. These polypeptides have been shown to beeffective in regulating the growth of cancer cells in vitro.Furthermore, the polypeptides were shown to elicit CD4+ T cell immunityagainst cancer cells harbouring the mutated p21-ras oncogene proteinthrough the administration of such polypeptides in vaccination or cancertherapy schemes. It has subsequently been shown that these polypeptidesalso elicit CD8+ T cell immunity against cancer cells harbouring themutated p21 ras oncogene protein through the administration mentionedabove (Gjertsen, M. K. et aL, 1997, Int. J Cancer 72: 784–790).

International Application PCT/NO99/00143 (published as WO99/58552)describes synthetic polypeptides and fragments of mutant proteinproducts arising from frameshift mutations occurring in genes in cancercells. These polypeptides correspond to, completely cover or arefragments of the processed frameshift mutant protein fragment aspresented by cancer cells or other antigen presenting cells, and arepresented as a HLA-polypeptide complex by at least one allele in everyindividual. In particular polypeptides resulting from frameshiftmutations in the BAX and hTGF□-RII genes are disclosed. Thesepolypeptides were shown to be effective in stimulating CD4+ and CD8+ Tcells in a specific manner.

However, the polypeptides described above will be useful only in certainnumbers of cancers involving oncogenes with point mutations, frameshiftmutations or translocation in a proto-oncogene or tumour suppressorgene. There is a strong need for an anticancer treatment or vaccine thatwill be effective against a generic range of cancers.

The concerted action of a combination of altered oncogenes andtumour-suppressor genes results in cellular transformation anddevelopment of a malignant phenotype. Such cells are however prone tosenescence and have a limited life-span. In most cancers,immortalisation of the tumour cells requires the turning on of an enzymecomplex called telomerase. In somatic cells, the catalytic subunit ofthe telomerase holoenzyme, hTERT (human telomerase reversetranscriptase), is not normally expressed. Additional events, such asthe action of proteins encoded by a tumour virus or demethylation ofsilenced (methylated) promoter sites, can result in expression of thegenes encoding the components of the functional telomerase complex intumour cells.

Due to the presence of telomerase in most types of cancer cells, theenzyme has been disclosed as a general cancer vaccine candidate(International Patent Application No. PCT/NO99/00220, published asWO00/02581). WO00/02581 describes a method for preventing or treatingcancer by generating a T cell response against telomerase-expressingcells in a mammal suffering (or likely to suffer from) cancer. It isdemonstrated in WO00/02581 that both CD4+ and CD8+ T cells can bestimulated by administration of polypeptides having sequences derivedfrom such a telomerase protein.

Alternative splice variants of the telomerase pre-mRNA have beenreported in the literature (Kilian, A. et al., 1997, Hum. Mol. Genet. 6:2011–2019). Kilian et al. (1997, supra) indicated that it was noteworthythat several splice variants were located with the critical RT (reversetranscriptase) domain of hTERT. They stated, however, that a fullunderstanding of the significance of the hTERT splice variants was notobtained and that further functional characterisation was required.

Analysis of the complete genomic sequence of the hTERT gene, hasverified that the different mRNA splice variants arise from the usage ofalternative splice sites in the hTERT pre-mRNA (Wick, M. et al., 1999,Gene 232: 97–106). Compared with the full-length hTERT mRNA, at leastfive additional splice variants have been detected. A schematic drawingof these variants are provided in FIG. 1, and FIG. 2 shows an alignmentof the proteins encoded. Two of the splice variants, named α-del (orDEL1) and β-del (or DEL2), represent deletions of specific codingsequences. The α-del variant has deleted the first 36 nucleotides ofexon 6 and encodes a protein which lacks a stretch of 12 internal aminoacids. In the β-del variant 182 nucleotides representing the entireexons 7 and 8 are missing, leading to a shift in the open reading frameand a truncated protein with a 44-amino acid long carboxyl terminus notpresent in the full-length hTERT protein. The remaining splice variantsresult from the use of alternative splice sites located inside intronregions, resulting in the insertion of intron sequences within the openreading frame and premature termination of translation. The σ-insert (orINS1) variant results from an insertion of the first 38 nucleotides ofintron 4. The σ-insert does not contain a stop codon, but instead, theopen reading frame extends 22 nucleotides into the normal sequence usingan alternative reading frame. The γ-insert (or INS3) variant is causedby insertion of the last 159 nucleotides from intron 14. Ins-4 containsthe first 600 nucleotides from intron 14 while at the same time havingdeleted exon 15 and most of exon 16. The truncated proteins resultingfrom translation of these splice variants are shown in FIG. 2.

Several recent studies have addressed the regulation of telomeraseactivity, and some correlation between hTERT mRNA transcription andtelomerase activity has been reported for several cell lines and tissues(Nakamura, T. M. et al., 1997, Science 277: 955–959; Meyerson, M. etal., 1997, Int. J. Cancer 85: 330–335; Nakayama, J. et al., 1998, NatureGenet. 18: 65–68; Liu, K. et al., 1999, Proc. Natl Acad. Sci. USA 96:5147–5152). Others studies have shown that telomerase activity isup-regulated through phosphorylation of the hTERT protein by proteinkinase Cα, and conversely, down-regulated by the presence of proteinkinase C inhibitors and phosphatase 2A (Li, H. et al., 1997, J. Biol.Chem. 272: 16729–16732; Li, H. et al., 1998, J. Biol. Chem. 273:33436–33442; Bodnar, A. G. et al., 1996, Exp. Cell Res. 228: 58–64; Ku,W. C. et al., 1997, Biochem. Biophys. Res. Comm. 241: 730–736).Alternative splicing of the hTERT pre-mRNA represents an additionalmechanism for regulating telomerase activity, and has been shown tomediate down-regulation during fetal kidney development and in adultovarian and uterine tissues (Ulaner, G. A. et al., 1998, Cancer Res. 58:4168–4172; Ulaner, G. A. et al., 2000, Int. J. Cancer 85: 330–335). Thefocus of the abovementioned studies has been on the α and β splicevariants, presumably because they delete sequences which are believed toencode critical reverse transcriptase motifs (Lingner, J. et al., 1997,Science 276: 561–567).

The present invention provides peptides and nucleic acids encoding saidpeptides based on the TERT γ and σ splice variants, and the novel use ofthese peptides and nucleic acids in medicine.

Thus according to the present invention there is provided a polypeptidefor use in medicine; wherein the polypeptide:

-   a) comprises a sequence given in SEQ ID NO: 1, 2, 3, 4, 5, 6 or 11;-   b) comprises 8 contiguous amino acids from SEQ ID NO: 1, 2, 3, 4, 5,    6 or 11, with the proviso that at least one of said 8 contiguous    amino acids is from SEQ ID NO: 1, 3, 5 or 11; or-   c) comprises 8 contiguous amino acids that have only one, two or    three amino acid changes (eg. substitutions) relative to the 8    contiguous amino acids as described in b) above, with the proviso    that that at least one of the 8 contiguous amino acids present is    from SEQ ID NO: 1, 3, 5 or 11;    wherein the polypeptide is capable of inducing a T cell response.

The term “comprises” used herein includes “consists”. The polypeptide(or nucleic acid) of the present invention may be flanked by one or moreamino acid (or nucleic acid) residues unless otherwise specified. Forexample, the polypeptide may be part of a fusion protein which has oneor more flanking domain at the N- or C-terminus to allow forpurification of the fusion protein.

Amino acid changes or modifications (eg. substitutions) in thepolypeptide may in particular be made to the anchor residues which fitinto HLA or MHC molecules for presentation to T cells. Enhanced bindingand immunogenic properties of the polypeptide to HLA or MHC moleculesmay thus be achieved (see Bristol, J. A. et al., 1998, J. Immunol.160(5): 2433–2441; Clay, T. M. et al., 1999, J. Immunol. 162(3):1749–1755).

The polypeptide described above optionally may:

-   a) have at least 55% sequence identity with a molecule comprising    the sequence of SEQ ID NO: 1, as determined by an NCBI BLASTP    Version 2.1.2 search with default parameters;-   b) have at least 55% sequence identity with a molecule comprising    the sequence of SEQ ID NO: 2, as determined by an NCBI BLASTP    Version 2.1.2 search with default parameters;-   c) have at least 40% sequence identity with a molecule comprising    the sequence of SEQ ID NO: 3, as determined by an NCBI BLASTP    Version 2.1.2 search with an Expect value of 1000 and other    parameters as default;-   d) have at least 40% sequence identity with a molecule comprising    the sequence of SEQ ID NO: 4, as determined by an NCBI BLASTP    Version 2.1.2 search with an Expect value of 1000 and other    parameters as default;-   e) have at least 70% sequence identity with a molecule comprising    the sequence of SEQ ID NO: 5, as determined by an NCBI BLASTP    Version 2.1.2 search with an Expect value of 100000 and other    parameters as default;-   f) have at least 50% sequence identity with a molecule comprising    the sequence of SEQ ID NO: 6, as determined by an NCBI BLASTP    Version 2.1.2 search with an Expect value of 10000 and other    parameters as default; or-   g) have at least 40% and preferably 60% sequence identity with a    molecule comprising the sequence of SEQ ID NO: 11, as determined by    an NCBI BLASTP Version 2.1.2 search with an Expect value of 1000 and    other parameters as default;

The NCBI BLASTP program can be found athttp://www.ncbi.nlm.nih.pov/blast/, and default parameters changed usingthe Advanced Search. Higher than default “Expect” values may be requiredwhen searching with small query sequences for matches to be displayed.The term “sequence identity” used herein refers to amino acid residuesin optimally aligned sequences which match exactly at correspondingrelative positions. For example, the NCBI BLASTP program provides apercentage value of identities between query and subject (“hit”)sequences.

The polypeptide described above may comprise a sequence as given in SEQID NO: 1, 2, 3, 4, 5, 6 or 11 or may be a fragment of a sequence asshown in SEQ ID NO: 1, 3, 5, 6 or 11.

While the polypeptides that are presented by HLA class II molecules areof varying length (12–25 amino acids), the polypeptides presented by HLAclass I molecules must normally be nine amino acid residues long inorder to fit into the class I HLA binding groove. A longer polypeptidewill not bind if it cannot be processed internally by an APC or targetcell, such as a cancer cell, before presenting in the class I restrictedHLA groove. Only a limited number of deviations from this requirement ofnine amino acids have been reported, and in those cases the length ofthe presented polypeptide has been either eight or ten amino acidresidues long. For reviews on polypeptide binding to MHC molecules seeRammensee, H.-G. et al. (1995) Immunogenetics 41: 178–228 and Barinaga(1992), Science 257: 880–881. Male, D. K. et al. (1996, AdvancedImmunology, Mosby, London) provide background information on the fieldof immunology.

The T cell response generated by the polypeptide described above may begenerated after intracellular cleavage of the polypeptide to provide afragment that fits into an MHC or HLA binding groove. Alternatively, thepolypeptide described above may not need intracellular cleavage to fitinto an MHC or HLA class I binding groove. In this case, the polypeptidemay be from 8 to 10 amino acids long. Also provided is a polypeptidedescribed above which does not need intracellular cleavage to fit intoan MHC or HLA class II binding groove. In this case, the polypeptide maybe from 12 to 25 amino acids long.

The T cell response according to the present invention may increase thenumber and/or activity of T helper and/or T cytotoxic cells.

Also provided is a polypeptide which does not stimulate a substantialcytotoxic T cell response in a patient against one or more of thefollowing: bone marrow stem cells, epithelial cells in colonic crypts orlymphocytes.

Further provided according to the present invention is a nucleic acidmolecule for use in medicine; wherein the nucleic acid molecule:

-   a) has a strand that encodes a polypeptide described above, as    described above;-   b) has a strand that is complementary with a strand as described    in a) above; or-   c) has a strand that hybridises with a molecule as described in a)    or b) above (eg. under stringent conditions).

Stringent hybridisation conditions are discussed in detail at pp1.101–1.110 and 11.45–11.61 of Sambrook, J. et al. (1989, MolecularCloning, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor). One example of hybridisation conditions that can be usedinvolves using a pre-washing solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA(pH 8.0) and attempting hybridisation overnight at 55° C. using 5×SSC.Hybridising nucleic acid sequences within the scope of the presentinvention include probes, primers or DNA fragments. The term primerincludes a single stranded oligonucleotide which acts as a point ofinitiation of template-directed DNA synthesis under appropriateconditions (eg. in the presence of four different nucleosidetriphosphates and an agent for polymerisation, such as DNA or RNApolymerase or reverse transcriptase) in an appropriate buffer and at asuitable temperature.

Also provided is a vector or cell for use in medicine comprising anucleic acid molecule according to the present invention.

Further provided is a binding agent for use in medicine; wherein thebinding agent binds to a polypeptide described above as described above.Said binding agent may be specific for a polypeptide as described above.Said binding agent may be an antibody or a fragment thereof. Saidbinding agent may be lectin.

The term antibody in its various grammatical forms is used herein torefer to immunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antibodycombining site or paratope. Such molecules are also referred to as“antigen binding fragments” of immunoglobulin molecules. Illustrativeantibody molecules are intact immunoglobulin molecules, substantiallyintact immunoglobulin molecules and those portions of an immunoglobulinmolecule that contain the paratope, including those portions known inthe art as Fab, Fab′, F(ab′)2 and F(v). Antibodies of the presentinvention may be monoclonal or polyclonal. The term antibody is alsointended to encompass single chain antibodies, chimeric, humanised orprimatised (CDR-grafted) antibodies and the like, as well as chimeric orCDR-grafted single chain antibodies, comprising portions from twodifferent species. For preparation of antibodies see Harlow, E. andLane, D. (1988, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor) and Harlow, E. and Lane, D. (1999,Using Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor). Immunological adjuvants for vaccinescomprising lecithin may be used to stimulate antibody production (seefor example U.S. Pat. No. 4,803,070).

Further provided according to the present invention is a T lymphocytefor use in medicine; wherein the T lymphocyte is capable of killing acell expressing a polypeptide described above according to the presentinvention or of helping in the killing of such a cell. Said T lymphocytemay be a T cytotoxic cell or a T helper cell.

Also provided is a clonal cell line for use in medicine comprising aplurality of T lymphocytes as described above. Also provided is amixture of T lymphocytes for use in medicine comprising a T helper cellor a clonal cell line of such cells and a T cytotoxic cell or a clonalcell line of such cells.

Also provided is a method of generating T lymphocytes capable ofrecognising and destroying tumour cells in a mammal, comprising taking asample of T lymphocytes from a mammal and culturing the T lymphocytesample in the presence of at least one polypeptide described above in anamount sufficient to generate hTERT γ-insert protein specific Tlymphocytes and/or hTERT σ-insert protein specific T lymphocytes.

Also provided is a B lymphocyte which may be useful in generatingantibodies according to the present invention. Hybridomas which arecapable of generating antibodies according to the present invention arealso included (see for example Koehler et al., 1975, Nature 256:495–497; Kosbor et al., 1983, Immunol. Today 4: 72; Cote et al., 1983,PNAS USA 80: 2026–2030; Cole et al., 1985, Monoclonal Antibodies andCancer Therapy, Alan R. Liss Inc., New York, pp. 77–96).

Further provided according to the present invention is the use of apolypeptide as described above, a nucleic acid as described above, avector or cell as described above, a binding agent as described above, aT lymphocyte as described above, a cell line as described above, or amixture of T lymphocytes as described above, in the preparation of amedicament for treating cancer, or in the preparation of a diagnosticfor diagnosing cancer. The cancer may be a mammalian cancer. Inparticular, the cancer may be human cancer. For example, the cancer maybe breast cancer, prostate cancer, pancreatic cancer, colo-rectalcancer, lung cancer, malignant melanoma, leukaemia, lymphoma, ovariancancer, cervical cancer or a biliary tract carcinoma.

Said medicament may be a vaccine.

The polypeptides described here are particularly suited for use in avaccine capable of safely eliciting either CD4+ or CD8+ T cell immunity.As the polypeptides may be synthetically produced, medicaments includingthe polypeptides do not include transforming cancer genes or other sitesor materials which might produce deleterious effects. The polypeptidesmay be targeted for a particular type of T cell response without theside effects of other unwanted responses.

Said medicament may be an antisense molecule or is capable of generatingan antisense molecule in vivo.

Said diagnostic may be provided in a kit. The kit may comprise means forgenerating a detectable signal (eg. a fluorescent label, a radioactivelabel) or a detectable change (eg. an enzyme-catalysed change). The kitmay include instructions for use in diagnosing cancer.

Further provided is a pharmaceutical composition comprising apolypeptide as described above, a nucleic acid as described above, avector or cell as described above, a binding agent as described above, aT lymphocyte as described above, a cell line as described above, or amixture of T lymphocytes as described above.

Said pharmaceutical composition may comprise a polypeptide capable ofinducing a T cell response directed against a polypeptide produced by anoncogene or against a mutant tumour suppressor protein, or a nucleicacid encoding such a polypeptide, or a binding agent that binds such apolypeptide, or a T cell that is capable of killing a cell expressingsuch a polypeptide or of helping in the killing of such a cell. Exampleof such oncogenes or mutant tumour suppressor proteins include p21-ras,Rb, p53, abl, gip, gsp, ret or trk. The oncogene target may be thep21-ras polypeptides described in International Application No.PCT/NO92/00032 (Publication No. WO92/14756).

Also provided is a combined preparation comprising a component from thepharmaceutical compositions described above for simultaneous, separateor sequential use in anticancer therapy.

Also provided is a pharmaceutical composition or a combined preparationas described above further comprising a pharmaceutically acceptablecarrier, diluent, additive, stabiliser, and/or adjuvant; saidcomposition or combined preparation optionally further including one ormore of: a cytokine or growth factor (eg. IL-2, IL-12; and/or GM-CSF)and another polypeptide arising from a frameshift mutation (eg. aframeshift mutation in the BAX or hTGFβ-RII gene.)

The stimulatory effect on CD4+ and CD8+ T cells in a specific manner bypolypeptides resulting from frameshift mutations in the BAX andhTGFβ-RII genes was disclosed in WO99/58552 (see above).

The pharmaceutical composition or combined preparation described abovemay be a vaccine.

The pharmaceutical composition or combined preparation described abovemay comprise or be capable of producing antisense molecules.

Also provided is a method for the preparation of a pharmaceuticalcomposition as described above, comprising the steps of combining theabove described components with a pharmaceutically acceptable carrier,diluent, additive, stabiliser and/or adjuvant.

A pharmaceutical composition according to the present may comprise anyof the following mixtures:

-   -   a) a mixture of at least one polypeptide described above        together with another polypeptide having a different sequence;    -   b) a mixture of at least one polypeptide described above        together with another polypeptide having an overlapping        sequence, so that the polypeptides are suitable to fit different        MHC or HLA alleles;    -   c) a mixture of both mixtures a) and b);    -   d) a mixture or several mixtures a);    -   e) a mixture of several mixtures b); or    -   f) a mixture of several mixtures a) and several mixtures b).

The polypeptides in the mixture may be covalently linked with each otherto form larger polypeptides or even cyclic polypeptides. Thepolypeptides themselves may be in a linear or cyclic form.

Also provided according to the present invention is a diagnosticcomposition comprising a polypeptide as described above, a nucleic acidas described above, a vector or cell as described above, a binding agentas described above, a T lymphocyte as described above, a cell line asdescribed above, or a mixture of T lymphocytes above.

Also provided according to the present invention is a diagnostic kit asdescribed above.

Also provided according to the present invention is a method oftreatment or prophylaxis of cancer of the human or animal bodycomprising administering a therapeutically effective amount ofpharmaceutical composition described above to a patient or animal inneed of same. The invention includes a method of treatment orprophylaxis of patient or animal afflicted with cancer, the methodcomprising administering to said patient or animal a pharmaceuticalcomposition described above in an amount sufficient to elicit a T-cellresponse against said cancer. The method of treatment may also includestimulation in vivo or ex vivo with a pharmaceutical compositiondescribed above. Ex vivo therapy may include isolating dendritic cellsor other suitable antigen presenting cells from a patient or animal,loading said cells with at least one polypeptide or nucleic aciddescribed above, and infusing these loaded cells back into the patientor animal. The polypeptides or nucleic acids described above may also beused in a method of vaccination of a patient in order to obtainresistance against cancer. Oncogenes are sometimes associated withviruses. The present invention is also suitable for the treatment ofcertain viral disorders.

The polypeptides according to the present invention may be administeredin an amount in the range of 1 microgram (1 μg) to 1-gram (1 g) to anaverage human patient or individual to be vaccinated. It is preferred touse a smaller dose in the range of 1 microgram (1 μg) to 1 milligram (1mg) for each administration.

The exact dosages, ie. pharmaceutically acceptable dosages, andadministration regime of pharmaceutical compositions and medicaments ofthe present invention may be readily determined by one skilled in theart, for example by using for example dose-response assays.

The administration may take place one or several times as suitable toestablish and/or maintain the desired T cell immunity. The polypeptidesaccording to the present invention may be administered together, eithersimultaneously or separately, with compounds such as cytokines and/orgrowth factors, i.e., interleukin-2 (IL-2), interleukin-12 (IL-12),granulocyte macrophage colony stimulating factor (GM-CSF) or the like inorder to strengthen the immune response as known in the art. Thepolypeptides can be used in a vaccine or a therapeutic compositioneither alone or in combination with other materials. For example, thepolypeptide or polypeptides may be supplied in the form of a lipopeptideconjugate which is known to induce a high-affinity cytotoxic T cellresponse (Deres, K. et al., 1989, Nature 342: 561–564).

The polypeptides according to the present invention may be administeredto an individual or animal in the form of DNA vaccines. The DNA encodingthe polypeptide(s) may be in the form of cloned plasmid DNA or syntheticoligonucleotide. The DNA may be delivered together with cytokines, suchas IL-2, and/or other co-stimulatory molecules. The cytokines and/orco-stimulatory molecules may themselves be delivered in the form ofplasmid or oligonucleotide DNA.

Response to a DNA vaccine has been shown to be increased by the presenceof immunostimulatory DNA sequences (ISS). These can take the form ofhexameric motifs containing methylated CpG, according to the formula:5′-purine-purine-CG-pyrimidine-pyrimidine-3′. DNA vaccines according tothe present invention may therefore incorporate these or other ISS, inthe DNA encoding the hTERT γ-insert protein and/or the hTERT σ-insertprotein, in the DNA encoding the cytokine or other co-stimulatorymolecules, or in both. A review of the advantages of DNA vaccination isprovided by Tighe et al. (1998, Immunology Today, 19(2): 89–97).

Also provided according to the present invention is the polypeptide asdescribed above, optionally in isolated form, wherein the polypeptide isnot a polypeptide consisting of the sequences shown in FIG. 4.

The polypeptide sequence shown in FIG. 4 represents the disclosure inFIG. 5C of Kilian et al. (1997, supra) of 46 amino acid residues at theC-terminal end of the circa 1100 amino acid residue hTERT γ-insertsplice variant, which includes 44 amino acids of SEQ ID NO: 1. Thesequence provided in Kilian et al. (1997, supra) shows the “alternativeC-terminus” of the hTERT γ-insert splice variant protein. (Kilian etal., 1997, supra, indicate that the corresponding DNA sequence inprovided by GenBank Accession number AF015950.) Kilian et al. (1997,supra) do not disclose as a separate entity the polypeptide according toSEQ ID NOs: 1, 2 or 5 at the C-terminal end of the hTERT γ-insert splicevariant protein, and they do not disclose or suggest medicinal use ofthe polypeptide according to SEQ ID NO: 1, 2 or 5.

The polypeptides described herein may be produced by conventionalprocesses, for example, by the various polypeptide synthesis methodsknown in the art. Alternatively, they may be fragments of a hTERTγ-insert protein and/or a hTERT σ-insert protein produced by cleavage,for example, using cyanogen bromide, and subsequent purification.Enzymatic cleavage may also be used. The hTERT γ-insert protein and thehTERT σ-insert protein or peptides may also be in the form ofrecombinant expressed proteins or polypeptides.

Also provided is the nucleic acid as described herein, optionally inisolated form; wherein the nucleic acid is not a nucleic acid encodingthe polypeptide excluded above and is also not a nucleic acid as shownin FIG. 4 or FIG. 5.

The nucleic acid sequence at the 3′-end of the circa 3100 bp hTERTγ-insert splice variant, part of which encodes the C-terminal end of thecorresponding protein that includes SEQ ID NO: 1, is provided in FIG. 4of Kilian et al. (1997, supra). The nucleic acid sequence at theexon-intron borders of the hTERT splice variants INS1 (equivalent to theσ-insert splice variant) and INS3 (equivalent to the γ-insert splicevariant), as disclosed in FIG. 2B of Wick et al. (1999, supra), areshown in FIG. 5. The nucleic acids shown in FIG. 5 as disclosed in FIG.2B of Wick et al. (1999, supra) include nucleotides which encode aminoacid residues present in SEQ ID NOs: 1–6 and 11. Wick et al. (1999,supra) make no specific reference to the existence of the nucleic acidsshown as distinct entities or to their medical use. Wick et al., 1999,supra, provide reference to the complete nucleotide sequence of theirhTERT gene in GenBank Accession numbers AF128893 and AF128894.

Nucleic acids encoding the polypeptides of the present invention may bemade by oligonucleotide synthesis. This may be done by any of thevarious methods available in the art. A nucleic acid encoding telomeraseprotein may be cloned from a genomic or cDNA library, using conventionallibrary screening. The probe may correspond to a portion of any sequenceof a known hTERT γ-insert and/or hTERT σ-insert gene. Alternatively, thenucleic acid can be obtained by using the Polymerase Chain Reaction(PCR). The nucleic acid is preferably DNA, and may suitably be clonedinto a vector. Subclones may be generated by using suitable restrictionenzymes. The cloned or subcloned DNA may be propagated in a suitablehost, for example a bacterial host. Alternatively, the host can be aeukaryotic organism, such as yeast or baculovirus. The hTERT γ-insertand the hTERT σ-insert proteins or polypeptides may be produced byexpression in a suitable host. In this case, the DNA is cloned into anexpression vector. A variety of commercial expression kits areavailable. The methods described in Sambrook, J. et al. (1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor) may be used for these purposes.

Also provided is the vector or cell as described herein, optionally inisolated form.

Further provided is the binding agent as described herein, optionally inisolated form.

Yet further provided is the T lymphocyte as described herein, optionallyin isolated form.

Also provided is the clonal cell line as described herein, optionally inisolated form.

Further provided is the mixture of T lymphocytes as described herein.

Also provided is a machine readable data carrier (eg. a disk) comprisingthe sequence of a polypeptide or of a nucleic acid as described herein.

Yet further provided is a method comprising using the sequence of apolypeptide or a nucleic acid molecule as described herein to performsequence identity studies, sequence homology studies, or hybridisationstudies. Said method may include using said sequence to predictstructure and/or function (eg. to predict anti-cancer activity). Alsoprovided is the use of this method in a drug development or screeningprocedure. Further provided is a drug identified or selected by thisprocedure.

Also provided is a computer or database that displays or stores asequence of a polypeptide or a nucleic acid molecule as described hereinor that is set up to perform a method as described above.

Also provided is the invention as substantially hereinbefore describedwith reference to the accompanying figures and examples.

The phrases “amino acid residue” and “amino acid” are broadly defined toinclude modified and unusual amino acids as defined in WIPO StandardST.25, and incorporated herein by reference.

The term treatment or therapy used herein includes prophylactictreatment or therapy where applicable.

The contents of each of the references discussed herein, including thereferences cited therein, are herein incorporated by reference in theirentirety.

The invention will be further apparent from the following description,with reference to the several accompanying figures, which show, by wayof example only, various polypeptides and their use according to thepresent invention.

Of the figures:

FIG. 1 is a schematic drawing of the full-length hTERT mRNA and splicevariants found in cancer cell lines;

FIG. 2 shows a protein alignment between a portion of the hTERT proteinand proteins resulting from translation of splice variants;

FIG. 3 shows the carboxyl termini of the hTERT γ-insert and σ-insertsplice variant proteins;

FIG. 4 shows prior art sequences relating to the hTERT γ-insert splicevariant as disclosed in FIG. 5C of Kilian et al. (1997, supra);

FIG. 5 show prior art sequences relating to the hTERT γ-insert andσ-insert splice variants as disclosed in FIG. 2B of Wick et al. (1999,supra);

FIG. 6 shows results from RT-PCR analysis of the regions comprising theγ-insert (A) and σ-insert (B) splice variants of hTERT;

FIG. 7 shows proliferative T cell responses induced in human bloodsamples by a polypeptide having the amino acid sequence of SEQ ID NO:11;

FIG. 8 shows proliferation of T cell clones induced by a polypeptidehaving the amino acid sequence of SEQ ID NO:11; and

FIG. 9 shows proliferation of other T cell clones induced by apolypeptide having the amino acid sequence of SEQ ID NO:11.

In FIG. 1, the position of introns present in the hTERT pre-mRNA isindicated by the letter “i” followed by an appropriate number. Insertionand deletion variants are shown as square boxes; shaded fill representssequences that encode protein sequence not present in the full-lengthhTERT protein. Position and orientation of oligonucleotide primers usedto analyse the different splice variants is indicated by arrows.

In FIG. 2, amino acid numbering is shown above the sequence. Amino acidsare represented by their standard one letter abbreviation known in theart.

In FIG. 3, SEQ ID NO: 1 reflects the truncated tail of the hTERTγ-insert protein and SEQ ID NO:2 reflects the same polypeptide with anextension at the amino terminus with the nine amino acids normally foundin these positions in the naturally occurring hTERT γ-insert expressionproduct (underlined). SEQ ID NO: 3 reflects the truncated tail of thehTERT σ-insert protein. SEQ ID NO: 4 reflects SEQ ID NO: 3 with anextension at the amino terminus with the nine amino acids normally foundin these positions in the naturally occurring hTERT σ-insert expressionproduct (underlined).

In FIG. 4, the exon/intron junctions of insert splice variant 3(equivalent to the hTERT γ-insert splice variant) is shown as providedin FIG. 5C of Kilian et al. (1997, supra). The following information isprovided by Kilian et al. (1997, supra): the nucleic acid sequence isshown above a protein translation sequence, with the putative unsplicedintron given in bold type; putative exon/intron junctions are markedwith |; the nucleic acid sequence numbering corresponds as follows:nucleotide 1 corresponds to nucleotide 139 of the sequence in GenBankAccession number AF015950; and amino acids corresponding to the putativec-Abl/SH3 binding site are underlined. The amino acid sequence shown byKilian et al. (1997; supra) represents the C-terminal end of a circa1100 amino acid residue hTERT γ-insert splice variant protein.

In FIG. 5, nucleotides of the exon-intron borders of the hTERT splicevariants INS1 equivalent to the σ-insert splice variant) and INS3(equivalent to the γ-insert splice variant), as disclosed in FIG. 2B ofWick et al. (1999, supra), are represented. Intronic and exonicsequences are shown in lower-case and upper-case letters, respectively.Wick et al. (1999, supra) indicate that the nucleotide sequence of theirhTERT gene has been deposited as GenBank Accession numbers AF128893 andAF128894.

It has been established that the hTERT γ-insert and σ-insert splicevariants are expressed in cancer cell lines and tumours but areundetectable, or present at very low levels, in normal cells. Thepresent application therefore discloses general cancer vaccinecandidates with improved specificity in comparison with vaccines basedon the functional variant of the telomerase (hTERT) protein. Both γ andσ inserts results in formation of an early stop codon and the expressionof a protein that is truncated at the carboxyl terminus. The truncatedhTERT γ- and σ-insert proteins have no telomerase activity themselves.In the case of a γ-insert the truncated tail of the protein is asequence of 44 amino acids (SEQ ID NO: 1). A σ-insert results in aprotein in which the truncated tail is a sequence of 20 amino acids (SEQID NO: 3). They are predominantly expressed in cancer cell lines but areundetectable, or present at very low levels, in normal cells and aretherefore targets for specific immunotherapy. According to the presentinvention, polypeptides corresponding to the carboxyl end (truncatedtail) of proteins expressed by hTERT γ-insert and/or σ-insert splicevariants, are useful as anticancer agents or vaccines with the functionto trigger the cellular arm of the immune system (T-cells) in humansagainst cancer cells. In a preferred embodiment of the invention, thepolypeptide comprises the sequence according to SEQ ID NO: 5. In anotherpreferred embodiment of the invention, the polypeptide comprises thesequence according to SEQ ID NO: 6. It yet another preferred embodiment,the polypeptide comprises the sequence according to SEQ ID NO: 11.

EXPERIMENTAL

The experiments outlined herein describe the characterisation of hTERTsplice variants in various cancer cell lines compared with normal cells.Synthesis of polypeptides according to the present invention, andexperiments for testing the efficacy of the polypeptides for use incancer therapy are detailed. An experiment showing induction andproliferation of human T cells by the peptide having an amino acidsequence according to SEQ ID NO: 11 is described.

RT-PCR Analysis of the γ-insert and σ-insert Splice Variants of hTERT

RNA Analysis:

Poly(A)⁺ mRNA from completely lysed cells was isolated directly fromcrude lysates using magnetic oligo(dT) beads (Dynal A S; Sakobsen, K. S.et al., 1990, Nucleic Acids Res. 18: 3669). Cytosolic mRNA fractionswere prepared by incubating cells in 1% IGEPAL (Sigma) at 0° C. for oneminute, followed by centrifugation [1000 g; 1 min.; 4° C.] to removenuclei. Poly(A)⁺ mRNA was then isolated from the supernatant usingoligo(dT) beads as described above.

cDNA Synthesis and PCR:

First strand cDNA synthesis was carried out by standard procedures usingM-MLV RNaseH÷ reverse transcriptase (Promega Corp.), and the PCRreactions were performed by using HotStar Taq DNA polymerase (Qiagen)and run for 35 cycles on a PTC-200 thermal cycler (MJ Research). Toobtain detectable products from PBM and CD34+ cells, 10% of the reactionwas used as template in a second PCR reaction and amplified by 15additional cycles.

For analysis of the γ-insert splice variant the plus-strand primervariant the plus-strand primer hTERT-p3195 (5-GCC TCC CTC TGC TAC TCCATC CT—SEQ ID NO: 7) and minus-strand primer hTERT-m3652 (5-CGT CTA GAGCCG GAC ACT CAG CCT TCA—SEQ ID NO: 8) were used. Applied on thefull-length hTERT cDNA and the γ-insert variant, these primers producefragments of 465 and 624 nucleotides, respectively. The analysis of theσ-insert variant was performed by using primers hTERT-P6 (5-GCC AAG TTCCTG CAC TGG CTG A—SEQ ID NO: 9) and hTERT-m2044 (5-GCT CTA GAA CAG TGCCTT CAC CCT CG—SEQ ID NO: 10). The amplification product resulting fromusing these primers with full-length hTERT cDNA and the σ-insert variantcomprises 369 and 407 nucleotides, respectively. To verify that thesePCR products represent genuine splice variants, the fragments wereisolated from the gel and analysed by direct sequencing using an ABIprism 310 automated sequencer (PE Corp.).

Results:

Telomerase activity is subject to complex regulation at thepost-transcriptional level, and methods used to detect the presence orabsence of telomerase proteins should involve direct measurements of theprotein itself, or alternatively, mRNA variants. Furthermore, theabundance of the different hTERT splice variants found in cells is notnecessarily correlated with the levels found in the cytosolic fractionof the same cells (see FIG. 6). Such deviations may be explained bydifferences in the efficiency with which mRNA variants are transportedfrom the nucleus to the cytosolic compartment, and/or by differentialstability of the specific splice variants in the cytosol. It is wellknown in the art that such mechanisms are part of the concept of generegulation. Nevertheless, the studies conducted to explain hTERTregulation, including those cited above, have used total RNA or mRNAisolated from completely lysed cells for their analysis. Kits andreagents required to perform this kind of RNA isolation are widelyavailable in the commercial market. To obtain a correct picture of geneexpression, studies on mRNA abundance should include analysis of mRNAspecific to the cytosolic compartment.

FIG. 6 shows results from RT-PCR analysis of the regions comprising theγ-(A) and σ-insert variants (B) of hTERT. HL60, K562, and Jurkat denotethe cancer cell lines analysed. HL60 is a promyelocytic leukemia cellline (Sokoloski, J. A. et al., 1993, Blood 82: 625–632), K562 anerythroid leukemia cell line (Lozzio, C. B. et al., 1975, Blood 45:321–324), while Jurkat is derived from acute T-lymphocyte leukemia cells(Gillis, S. et al., 1980, J. Exp. Med. 152: 1709–1719). The HL60, K562,and Jurkat cancer cell lines are commercially available (for example,from ATCC, Oslo). PBM1, PBM2, PBM3 and PBM4 represent peripheral bloodmononuclear (PBM) cell populations isolated from four different healthydonors. CD34 denotes CD34-positive stem cells isolated from a healthydonor, and CC1/CC2 denotes colon cancer biopsies obtained from twocancer patients at the Norwegian Radium Hospital, Oslo, with CC2a andCC2b being two tissue samples dissected from the same tumour. RT-PCRreactions performed with mRNA isolated by complete lysis of cells andwith mRNA isolated from cytosolic fractions are marked with the letters“T” and “C”, respectively. “M” indicates lane with molecular weightmarker. Position of PCR fragments representing the γ- and σ-insertsplice variants and the respective full-length hTERT products (+) isindicated on the right side of the panels.

The RT-PCR analysis showed that both γ- and σ-insert splice variantswere readily detectable in all cancer cell lines and in one of thetumour samples analysed (CC2b), and with the σ-insert variant appearingas the most abundant in cytosolic fractions. In contrast, we were notable to detect these variants in cytosolic mRNA populations isolatedfrom PBM cells despite the extensive PCR amplification performed withthese samples. The identity of the weak 395-bp fragment produced withthe σ-insert primers on PBM and CD34-positive cells is at presentunknown.

Polypeptide Synthesis and Analysis for Applications Relating to Cancer

Polypeptide Synthesis:

The polypeptides were synthesised by using continuous flow solid phasepeptide synthesis. N-a-Fmoc-amino acids with appropriate side chainprotection were used. The Fmoc-amino acids were activated for couplingas pentafluorophenyl esters or by using either TBTU or diisopropylcarbodiimide activation prior to coupling. 20% piperidine in DMF wasused for selective removal of Fmoc after each coupling. Cleavage fromthe resin and final removal of side chain protection was performed by95% TFA containing appropriate scavengers. The polypeptides werepurified and analysed by reversed phase HPLC. The identity of thepolypeptides was confirmed by using electro-spray mass spectroscopy.

Polypeptide Testing and Cancer Therapy:

In order for a cancer vaccine according to the present invention, andmethods for specific cancer therapy based on T cell immunity to beeffective, two conditions must be met:

-   (a) the polypeptide is at least 8 amino acids long and is a fragment    of the hTERT γ-insert protein or the hTERT σ-insert protein and-   (b) the polypeptide is capable of inducing, either in its full    length or after processing by antigen presenting cell, T cell    responses.

The following experimental methods may be used to determine if these twoconditions are met for a particular polypeptide. First, it should bedetermined if the particular polypeptide gives rise to T cell immuneresponses in vitro. It will also need to be established if the syntheticpolypeptides correspond to, or are capable after processing to yield,polypeptide fragments corresponding to polypeptide fragments occurringin cancer cells harbouring the hTERT γ-insert protein and/or the hTERTσ-insert protein or antigen presenting cells that have processednaturally occurring hTERT γ-insert protein and/or hTERT σ-insertprotein. The specificity of T cells induced in vivo by hTERT γ-insertand/or hTERT σ-insert polypeptide vaccination may also be determined.

In Vitro T Cell Response Analysis;

It is necessary to determine if hTERT γ-insert and/or hTERT σ-insertexpressing tumour cell lines can be killed by T cell clones obtainedfrom peripheral blood from carcinoma patients after hTERT γ-insertand/or hTERT σ-insert polypeptide vaccination. T cell clones areobtained after cloning of T-cell blasts present in peripheral bloodmononuclear cells (PBMC) from a carcinoma patient after hTERT γ-insertand/or hTERT σ-insert polypeptide vaccination. The polypeptidevaccination protocol includes several in vivo injections of polypeptidesintracutaneously with GM-CSF or another commonly used adjuvant. Cloningof T cells is performed by plating responding T cell blasts at 5 blastsper well onto Terasaki plates. Each well contains 2×10⁴ autologous,irradiated (30 Gy) PBMC as feeder cells. The cells are propagated withthe candidate hTERT γ-insert and/or hTERT σ-insert polypeptide at 25 μMand 5 U/ml recombinant interleukin-2 (rIL-2) (Amersham, Aylesbury, UK)in a total volume of 20 ml. After 9 days T cell clones are transferredonto flat-bottomed 96-well plates (Costar, Cambridge, Mass.) with 1mg/ml phytohemagglutinin (PHA, Wellcome, Dartford, UK), 5 U/ml rIL-2 andallogenic irradiated (30 Gy) PBMC (2×10⁵) per well as feeder cells.Growing clones are further expanded in 24-well plates with PHA/rIL-2 and1×10⁶ allogenic, irradiated PBMC as feeder cells and screened forpolypeptide specificity after 4 to 7 days.

T cell clones are selected for further characterisation. Thecell-surface phenotype of the T cell clone is determined to ascertain ifthe T cell clone is CD4+ or CD8+. T cell clone is incubated withautologous tumour cell targets at different effector to target ratios todetermine if lysis of tumour cells occurs. Lysis indicates that the Tcell has reactivity directed against a tumour derived antigen, forexample, hTERT γ-insert and/or hTERT σ-insert proteins.

Correlation between Polypeptides and in vivo hTERT Insert Fragments;

In order to verify that the antigen recognised is associated with hTERTγ-insert protein or hTERT σ-insert protein, and to identify the HLAclass I or class II molecule presenting the putative hTERT γ-insert orhTERT σ-insert polypeptide to the T cell clone, different hTERT γ-insertand/or hTERT σ-insert expressing tumour cell lines carrying one or moreHLA class I or II molecules in common with those of the patient, areused as target cells in cytotoxicity assays. Target cells are labelledwith ⁵¹Cr or ³H-thymidine (9.25×10⁴ Bq/mL) overnight, washed once andplated at 5000 cells per well in 96 well plates. T cells are added atdifferent effector to target ratios and the plates are incubated for 4hours at 37° C. and then harvested before counting in a liquidscintillation counter (Packard Topcount). For example, the bladdercarcinoma cell line T24 (12Val⁺, HLA-A1⁺, B35⁺), the melanoma cell lineFMEX (12Val⁺, HLA-A2⁺, B35⁺) and the colon carcinoma cell line SW 480(12Val⁺, HLA-A2⁺, B8⁺) or any other telomerase positive tumour cell linemay be used as target cells. A suitable cell line which does not expresshTERT γ-insert and/or hTERT σ-insert proteins may be used as a control,and should not be lysed. Lysis of a particular cell line indicates thatthe T cell clone being tested recognises an endogenously-processed hTERTγ-insert and/or hTERT σ-insert epitope in the context of the HLA class Ior class II subtype expressed by that cell line.

Characterisation of T Cell Clones:

The HLA class I or class II restriction of a T cell clone may bedetermined by blocking experiments. Monoclonal antibodies against HLAclass I antigens, for example the panreactive HLA class I monoclonalantibody W6/32, or against class II antigens, for example, monoclonalsdirected against HLA class II DR, DQ and DP antigens (B8/11, SPV-L3 andB7/21), may be used. The T cell clone activity against the autologoustumour cell line is evaluated using monoclonal antibodies directedagainst HLA class I and class II molecules at a final concentration of10 μg/ml. Assays are set up as described above in triplicate in 96 wellplates and the target cells are preincubated for 30 minutes at 37° C.before addition of T cells.

The fine specificity of a T cell clone may be determined usingpolypeptide pulsing experiments. To identify the hTERT γ-insert and/orhTERT σ-insert polypeptide actually being recognised by a T cell clone,a panel of nonamer polypeptides is tested. ⁵¹Cr or ³H-thymidinelabelled, mild acid eluted autologous fibroblasts are plated at 2500cells per well in 96 well plates and pulsed with the polypeptides at aconcentration of 1 μM together with b2-microglobulin (2.5 μg/mL) in a 5%CO₂ incubator at 37° C. before addition of the T cells. Assays are setup in triplicate in 96 well plates and incubated for 4 hours with aneffector to target ratio of 5 to 1. Controls can include T cell clonecultured alone, with APC in the absence of polypeptides or with anirrelevant melanoma associated polypeptide MART-1/Melan-A polypeptide.

An alternative protocol to determine the fine specificity of a T cellclone may also be used. In this alternative protocol, the TAP deficientT2 cell line is used as antigen presenting cells. This cell lineexpresses only small amounts of HLA-A2 antigen, but increased levels ofHLA class I antigens at the cell surface can be induced by addition ofb2-microglobulin. ³H-labelled target cells are incubated with thedifferent test polypeptides and control polypeptides at concentration of1 μM together with b2-microglobulin (2.5 μg/mL) for one hour at 37° C.After polypeptide pulsing, the target cells are washed extensively,counted and plated at 2500 cells per well in 96 well plates beforeaddition of the T cells. The plates are incubated for 4 hours at 37° C.in 5% CO₂ before harvesting. Controls include T cell clone culturedalone or with target cells in the absence of polypeptides. Assays wereset up in triplicate in 96 well plates with an effector to target ratioof 20 to 1.

The sensitivity of a T cell clone to a particular polypeptide identifiedabove may also be determined using a dose-response experiment.Polypeptide sensitised fibroblasts can be used as target cells. Thetarget cells are pulsed with the particular peptide as described abovefor fine specificity determination, with the exception that the peptidesare added at different concentrations before the addition of T cells.Controls include target cells alone and target cells pulsed with theirrelevant melanoma associated peptide Melan-A/Mart-1.

Induction and Proliferation of Human T Cell Response to the hTERTσ-insert Peptide

In this experiment, peripheral blood mononuclear cells (PBMC) from fourhealthy humans (donors “14328”, “14313”, “23244” and “23255”) and wereisolated and primed for seven days with dendritic cells pulsed with theSEQ ID NO: 11 peptide derived from the hTERT σ-insert polypeptide,followed by two cycles consisting of seven days re-stimulation withpeptide-pulsed autologous PBMC. The dendritic cells were derived frommonocytes from peripheral blood. T cells from the resulting bulk culturewere tested in triplicate with or without peptide-pulsed antigenpresenting cells (APC) before harvesting after 3 days. To measure theproliferative capacity of the cultures, ³H-thymidine (3.7×10⁴ Bq/well)was added to the culture overnight before harvesting. Cultures withnon-pulsed APC or without APC served as controls. The results showingthe proliferative capacity of the cultures are shown in FIG. 7. Furtherdetails of the protocol used are set out below.

T cell clones were obtained from the resulting bulk cultures fromnon-vaccinated donors 14313 and 23255. The clones were obtained from Tcell blasts preset in PRMCs as described in the above section “In vitroT cell response analysis”. The results of proliferation of the T cellclones with peptide-pulsed and non-peptide pulsed anitgen presentingcells are shown in FIG. 8 (donor 14313) and FIG. 9 (donor 23255).

Results in FIGS. 7, 8 and 9 are given as mean counts per minute (cpm) oftriplicate measurements. The data demonstrates that blood from humanscontain circulating T cells specific for a peptide (SEQ ID NO: 11)derived from the peptide derived from the hTERT σ-insert polypeptide,and furthermore that such T cells can be expanded in vitro followingstimulation with the relevant peptide.

Thus, the experiments of FIGS. 7, 8 and 9 show that the hTERT σ-insertpolypeptide is immunogenic in man. In vitro (or in vivo) stimulation canthis give rise to hTERT σ-insert protein-specific T cell responses withthe potential to recognise the same antigen when overexpressed by atumour growing in a cancer patient. This particular experimentdemonstrates that in principle the peptide of SEQ ID NO: 11 could bedeveloped as a cancer vaccine in humans.

Protocol for Induction of MHC Class II Restricted T Cell Response

Day 0:

PBMCs were separated out from 50 ml of blood (from buffy coat). Thecells were counted and re-suspended in complete RPMI-1640/15% poolserum.

Bulk cultures were set up with 1–2 wells on a 24-well plate of PBMCs at2×10⁶ cells/ml in 1–1.5 ml. 25 μM of SEQ ID NO:11 peptide derived fromthe hTERT σ-insert polypeptide were added.

Day 9–10:

Bulk cultures were harvested and stimulated with irradiated PBMCs andpeptide. If there was high cell death, cultures were lymphoprepseparated, otherwise they were counted and resuspended in RPMI/15% poolserum. (Lymphoprep centrifugation of bulk cultures is carried out in 15ml Falcon tubes by 1; adding 8 ml of cell suspension, and 2; underlaywith 2 ml of lymphoprep. Spin at 1500 rpm for 30 min, and wash twicewith salt water.) One vial of autologous PBMCs was defrosted, washed,counted and resuspended in RPMI/15% FCS. The PBMCs were irradiated (25GY, 5 min 58 sec). Cells were plated out in 24-well plates. 0.5–2.0×10⁶T cells from bulk cultures were stimulated with 1×10⁶ irradiated feedercells (PBMCs) and 25 μM SEQ ID NO:11 peptide. The final volume was 1 ml.

Day 12:

IL-2 (10 U/ml) was added. Medium was also added if necessary byreplacing half the volume. Cultures were split if necessary.

Day 17:

The T cells in bulk culture were re-stimulated as on day 10, withautologous, irradiated PBMC's and SEQ ID NO: 11 peptide.

Day 19:

IL-2 (10 U/ml) was added to day 17 re-stimulated bulk cultures.

Day 24:

A proliferation assay for testing T cells for peptide specificity wasset up in 96-well plates, each condition in triplicate:

Triplicates 1–3 4–6 7–9 10–12 Controls PBMC¹ Tc² PBMC¹ + PBMC¹ + Tc²Tc² + IL-2³ Test PBMC¹ + PBMC¹ + Tc² + samples Tc² + Pep⁴ Pep⁴ + IL-2³¹50 000 irradiated PBMCs, ²50 000 T cells from bulk culture, ³1 U/ml,⁴25 μg/ml SEQ ID NO: 11 peptide

On day 2–3 of proliferation assay, ³H-Thymidine (20 μl) was added, andincubated at 37 C overnight before harvesting.

In a variant of the protocol (as used in the above example) the PBMCswere, on Day 0, primed with dendritic cells pulsed with SEQ ID NO: 11.

1. A method of stimulating the proliferation of human T cells,comprising the steps of: (a) obtaining T cells from a human cancerpatient and (b) contacting the T cells obtained in step (a) with apolypeptide comprising a sequence given in SEQ ID NO: 3, 4, 5, 6 or 11or a fragment of at least 8 contiguous amino acids of the SEQ ID NO: 3,4, 5, 6 or 11 sequences.
 2. A method according to claim 1, wherein thepolypeptide is from 8 to 10 amino acids long.
 3. A method according toclaim 1, wherein the polypeptide is from 12 to 25 amino acids long.
 4. Amethod according to any one of claims 1 through 3, wherein the T cellresponse increases the number and/or activity of T helper and/or Tcytotoxic cells.
 5. An isolated polypeptide comprising the sequencegiven in SEQ ID NO
 3. 6. The isolated polypeptide of claim 5, whereinthe polypeptide contains a maximum of 25 amino acid residues.
 7. Theisolated polypeptide of claim 6, wherein the polypeptide consists of thesequence given in SEQ ID NO
 3. 8. A composition comprising an isolatedpolypeptide comprising the sequence given in SEQ ID NO 3 in admixturewith at least one of a diluent, an additive, a stabilizer, and anadjuvant.
 9. The composition according to claim 8, wherein the adjuvantis GM-CSF.
 10. The composition according to claim 8, wherein thepolypeptide contains a maximum of 25 amino acid residues.
 11. Thecomposition according to claim 10, wherein the polypeptide consists ofthe sequence given in SEQ ID NO
 3. 12. The composition according toclaim 10, wherein the polypeptide is in admixture with GM-CSF.
 13. Amethod of stimulating the proliferation of human T cells, comprising thesteps of: (a) obtaining T cells from a human cancer patient and (b)contacting the T cells obtained in step (a) with a polypeptidecomprising the sequence given in SEQ ID NO
 3. 14. The method accordingto claim 13, wherein the polypeptide contains a maximum of 25 amino acidresidues.
 15. The method according to claim 14, wherein the polypeptideconsists of the sequence given in SEQ ID NO
 3. 16. The isolatedpolypeptide of claim 5, wherein the polypeptide comprises the sequencegiven in SEQ ID NO:
 4. 17. The isolated polypeptide of claim 16, whereinthe polypeptide consists of the sequence given in SEQ ID NO:
 4. 18. Theisolated polypeptide of claim 5, wherein the polypeptide comprises thesequence given in SEQ ID NO:
 11. 19. The isolated polypeptide of claim18, wherein the polypeptide contains a maximum of 25 amino acidresidues.
 20. The isolated polypeptide of claim 19, wherein thepolypeptide consists of the sequence given in SEQ ID NO:
 11. 21. Thecomposition according to claim 9, wherein the polypeptide comprises thesequence given in SEQ ID NO:
 4. 22. The composition according to claim21, wherein the polypeptide consists of the sequence given in SEQ ID NO:4.
 23. The composition according to claim 9, wherein the polypeptidecomprises the sequence given in SEQ ID NO:
 11. 24. The compositionaccording to claim 23, wherein the polypeptide contains a maximum of 25amino acid residues.
 25. The composition according to claim 24, whereinthe polypeptide consists of the sequence given in SEQ ID NO:
 11. 26. Themethod according to claim 13, wherein the polypeptide comprises thesequence given in SEQ ID NO:
 4. 27. The method according to claim 26,wherein the polypeptide consists of the sequence given in SEQ ID NO: 4.28. The method according to claim 13, wherein the polypeptide comprisesthe sequence given in SEQ ID NO:
 11. 29. The method according to claim28, wherein the polypeptide contains a maximum of 25 amino acidresidues.
 30. The method according to claim 29, wherein the polypeptideconsists of the sequence given in SEQ ID NO: 11.